This project will study structure/function relationships of the myosin isoform found in muscle, myosin II. Myosin II is the protein that, through its interaction with actin, is the force generator of skeletal, cardiac and smooth muscle. The primary goal of this study is to define the domains within the myosin heavy chain that underlie the functional differences between myosin heavy chain isoforms. This project will focus on five regions, which include three surface loops (the 25/50 KDa junction, the 50/20 KDa junction and the secondary actin binding site), the amino terminal "knob" and the neck region of the myosin head. In addition, we will identify the structural elements that are involved in regulation (via reversible phosphorylation of the myosin light chain) of smooth muscle myosin. In order to study the myosin II heavy chain, we have developed a heterologous expression system. This entails myosin expression in a cultured cell line derived from insects (SF9 cells) that can be infected with a virus (baculovirus) altered in the laboratory so that it will produce large amounts of smooth muscle myosin. For all expressed recombinant S1 and HMM fragments, steady state ATPase activity (Vmax and K-ATPase) as well as actin binding (K-binding) will be determined. In addition, stopped flow measurements of S1 will allow determination of the rate of phosphate release, the rate of ADP release and the rate of myosin dissociation from actin. In vitro motility measurements will be performed on all HMM constructs. HMM will be characterized in both the phosphorylated and dephosphorylated states, whereas S1 assays will involve dephosphorylated protein. Three-dimensional reconstructions of cryo- electron microscopy of recombinant S1-decorated actin filaments will allow localization of structural elements. Thus we will characterize the impact of variations in the regions under investigation on the structure as well as the enzymatic properties of the myosin motor. In this manner we will begin to dissect the functional and structural domains of the myosin motor, within the framework of delineating the principles governing myosin heavy chain isoform diversity.